Oxidative stress is a key determinant in the development of diabetes related microvascular and cardiovascular complications. ShcA (Src homology and collagen homology) family proteins are prototypical signaling adaptors that regulate a diverse array of cellular response pathways (e.g., tyrosine kinase (TK) signaling). One ShcA family member, p66Shc, is also a major source (~30%) of reactive oxygen species (ROS) production in mammalian cells. ShcA-mediated ROS production is a determinant of stroke size in cardiac ischemia reperfusion injury and endothelial dysfunction in diabetes (e.g., delayed cutaneous wound healing) and hypertension. The molecular basis for ShcA enzymatic function is poorly defined or unknown. This project is focused on defining how ShcA proteins function at the molecular level using a range of biophysical methods. ShcA proteins have proven difficult to study in defined systems due to their low expression profile in standard lab systems, they are conformationally variable and prone to posttranslational modification, and they interact with a broad range of proteins in vivo. The overarching hypothesis of this project is that ShcA proteins function as molecular rheostats to modulate redox state, cyt c activity, and metabolic flux in a location, conformation, and binding-dependent manner. We will investigate this hypothesis by pursuing the following Specific Aims: 1) define the enzymatic mechanism of ShcA-mediated ROS production, 2) define the functional interactions between ShcA proteins and cytochrome c, and 3) determine the structural basis for ShcA-mediated function. Outflow from this project will provide a molecular basis for ShcA function as a means to facilitate downstream development of isoform specific inhibitors of ShcA-mediated ROS production. In addition, ShcA proteins function at the crossroads of cell survival and apoptosis so this project will further define the role that ShcA proteins play in determining cellular stress response.